The present invention relates to a detection apparatus for detecting the presence of a detectable material in a sample. More specifically, the present invention relates to a detection apparatus for detecting the presence of a biological component in urine, blood, or the like. Additionally, the present invention relates to a method for the detection of a biological component in urine, blood, or the like.
Among conventional detection methods for detecting a biological component detectable material, there is an immunological detection method, which uses antibodies and antigens. This method, which provides high sensitivity and good specificity and ease of use, is used widely in the field of clinical testing. Various technologies have been recently proposed to perform testing using the immunological detection method without requiring any special techniques or training.
Of these, a group of measuring methods known as immunological chromatography has been proposed (Japanese laid-open patent publication number 9-178748) and is already being marketed to the general population. This method provides quick, easy, and accurate results.
In immunological chromatography, a marked reagent (staining indicator) using colored latex, colloidal gold, or the like, is fixed to a spreading layer. An unmarked reagent is fixed to a detection zone on the spreading layer. When the detectable material is present in the fluid sample, immunoreaction compounds are produced. The staining marker from this reaction is caught so that it can be visually observed. With the immunological chromatography method, the fluid sample can be simply applied to an application position. After a fixed period of time, the degree of coloration from the staining marker is observed at the detection zone.
With this method, the antigens or antibodies which have affinity for the detectable material must be prepared along with enzymes, fluorescent materials, luminescent materials, colored latex, colloidal gold, or the like, which serve as markers, that are chemically or physically bonded with marker antigens or marker antibodies depending on the application. However, the marking reaction involves a major problem. For example, to bond an enzyme to an antigen or antibody protein, a chemical procedure such as periodic acid oxidation can be performed (Enzyme Immunoassay, Igaku-Shoin, Tokyo, 1982). In such cases, irreversible deactivation of the antigen, antibody, or the enzyme takes place. Additionally, polymerization leading to reduction of activation or non-specific reaction can take place. As a result, the practical marker yield becomes very low.
Similarly, when physical (hydrophobic bonding) methods are used to bond colored latex to antigen or antibody proteins, the bonding to the colored latex takes place randomly so that the active sites of the antigen or antibodies are lost. This provides inadequate reactions so that excess antigens or antibodies must be used. Also, since absorption does not proceed 100%, significant antigens or antibodies are wasted.
To overcome these problems, a method that uses bivalent reagents, as a crosslinking agent, has been developed (Enzyme Immunoassay, Igaku-Shoin, Tokyo, 1982). However, the operations involved are complex and require a high degree of skill. Furthermore, since the reaction itself is highly sensitive, slight changes in reaction conditions can greatly affect the properties of the marker. Thus, the bivalent reagent method of preparing markers is considered not very reproducible.
When using the conventional high-specificity marking method, the loss of activation of the markers or the antigens/antibodies or the like is unavoidable, and 100% marking cannot be achieved since the marking operation is itself a chemical reaction.
It is an object of the present invention to provide a detection method for minimizing reduced activation and antibody loss from the marking operation while providing measurement sensitivity that is at least equal to that of the conventional technology.
It is a further object of the present invention is to provide a detection apparatus that gives highly sensitive and inexpensive immunological detection without wasting valuable antibodies.
It is another object of the present invention to provide a detection method that gives highly sensitivity and inexpensive immunological detection without wasting valuable antibodies.
It is still a further object of the present invention to provide a high-sensitivity detection apparatus and method for detecting a detectable material that does not involve restrictions of reagent amounts.
Briefly stated, the present invention provides a catcher, having an immunological epitope, fixed to a detection zone of a spreading layer. A marker, capable of easy detection, has an immunological epitope. The marker and bispecific antibodies are soluble so that they can move on the spreading layer. The bispecific antibodies include a first bispecific antibody, having specificity for the detectable material in the fluid sample and the marker, and a second bispecific antibody, having specificity for the detectable material in the fluid sample and the catcher. Pore sizes and particle diameters are set so that the reaction product in which the marking elements and the particles are bonded are caught at a catching section. The concentration of the marking elements and the particles are increased to improve detection sensitivity. The result is an inexpensive and simple detection apparatus being highly-sensitive for the immunological detection of a detectable material, without wasting valuable antibodies.
In the conventional detection apparatus, as described above, the unmarked reagent is fixed to the detection zone on the spreading layer. With this structure, the amount of reagent is restricted by its ability to bond with protein. Thus, it is difficult to use more than a fixed amount. Also, in order to detect the detectable material, the detectable material must be biologically bonded to both the reagent component and the marking component. The restriction on the amount of reagent prevents the sensitivity from going beyond a certain point.
Furthermore, the reagent is fixed to the spreading layer. Therefore, out of the time that the detectable material is chromatographically moving along the spreading layer, the reaction can only take place during the very short interval of time when the detectable material contacts the reagent. This reduces the reaction and results in low reactivity in the conventional detection device. With regard to this problem, a technology has been proposed to use magnetism (Japanese laid-open patent publication number 5-52849), but this requires the use of materials having magnetism and therefore cannot be applied to a wide range of materials.
According to an embodiment of the present invention, there is provided a detection apparatus for detecting the presence of a detectable material in a sample comprising: a spreading layer on which the sample is applied; a catcher fixed to the spreading layer at a detection zone away from a position at which the sample is applied; the catcher including an immunological epitope; bispecific antibodies supported in the spreading layer in a dry state, such that movement of the bispecific antibodies is possible when the bispecific antibodies are in a soluble state; a marker supported in the spreading layer in a dry state, such that movement of the marker is possible when the marker is in a soluble state; the marker including an immunological epitope being capable of detection; the double-specific antibody including a first double-specific antibody and a second double-specific antibody; the first double-specific antibody being specific to the detectable material in the sample as well as the marker; and the second double-specific antibody being specific to the detectable material in the sample as well as the catcher.
According to another embodiment of the present invention, there is provided a detection method for detecting the presence of a detectable material in a sample comprising: applying the sample to one end of a spreading layer such that the sample chromatographically moves in a direction toward the other end of the spreading layer; solubilizing a first bispecific antibody, a second bispecific antibody, and a marker, thereby permitting movement of the first bispecific antibody, the second bispecific antibody, and the marker along the spreading layer; bonding the detectable material with the first bispecific antibody and the second bispecific antibody such that the detectable material is interposed therebetween; bonding the first bispecific antibody with the marker; bonding the second bispecific antibody with a catcher fixed to the spreading layer at a detection zone located a prescribed distance from a point where the sample was applied to the spreading layer; and analyzing presence of the marker at the detection zone, whereby the presence of the marker corresponds with presence of the detectable material.
According to a further embodiment of the present invention, there is provided a detection apparatus for detecting the presence of a detectable material in a sample comprising: a fluid application section contacting the sample; a reaction reagent section, having particles and marking elements movably contained therein, connected to the fluid application section such that the sample moves from the fluid application section to the reaction reagent section; a porous carrier connected to the reaction reagent section such that the sample moves from the reaction reagent section to the porous carrier; a reaction product formed from biologically bonding the detectable material with both the marking elements and the particles when the detectable material is present in the sample; and a catching section in the porous carrier made from a material having a pore size smaller than a size of the reaction product, such that chromatographic movement of the marking elements not bonded to the particles is permitted through said catching section and chromatographic movement of the reaction product is restricted.
According to still another embodiment of the present invention, there is provided a detection method for detecting the presence of a detectable material in a sample comprising: contacting the sample with a fluid application section; chromatographically moving the sample through the fluid application section, a reaction reagent section, and a porous carrier; reacting the sample with particles and marking elements contained in the reaction reagent section to form a reaction product, such that the detectable material bonds with both the marking elements and the particles when the detectable material is present in the sample; passing the sample, including any reaction product present, through a catching section having a pore size smaller than a size of the reaction product and larger than a pore size of the marking elements; and analyzing presence of the marking elements at the catching section, whereby presence of the marking elements corresponds to presence of the detectable material.
A detection apparatus according to the present invention includes a spreading layer onto which a fluid sample is applied. A detection zone is disposed on the spreading layer at a position away from the position at which the fluid sample is applied. A catcher containing an immunological epitope is fixed to the detection zone. A detectable marker contains an immunological epitope. The marker and bispecific antibodies are supported in a dry state in the spreading layer so that when soluble they can move. The bispecific antibodies contain a first bispecific antibody, which has specificity for the marker and the detectable material in the fluid sample, and a second bispecific antibody, which has specificity for the catcher and the detectable material in the fluid sample.
A detection apparatus according to the present invention includes a fluid application section placed in contact with the sample fluid, a reaction reagent section connected to the fluid application section, and a porous carrier connected to the reaction reagent section. The reaction reagent section contains particles, which do not affect detection, and marking elements which, through biochemical reactions, bond with the particles through the detectable material. The particles and the marking elements are able to move through the porous carrier. A catching section, disposed at the detection zone, prevents chromatographical movement of the reaction product produced from the bonding of the particles and the marking elements with the detectable material. The catching section also allows chromatographic movement of marking elements not bonded with particles. The pore size of the catching section is smaller than the particle diameter of the reaction product and is larger than the particle diameter of the marking elements not bonded with particles.
The detection apparatus according to the present invention includes a spreading layer on which a fluid sample is applied. A catcher is fixed to the spreading layer at a detection zone away from a position at which the fluid sample is applied. The catcher includes an immunological epitope. Bispecific antibodies and a marker are supported in the spreading layer in a dry state so that movement is possible when in a soluble state. The marker includes an immunological epitope and is capable of being detected. The bispecific antibodies include a first bispecific antibody and a second bispecific antibody. The first bispecific antibody is specific to a detectable material in the fluid sample as well as the marker. The second bispecific antibody is specific to the detectable material in the fluid sample as well as the catcher.
With this structure, detection is performed without the use of marker antibodies. Thus, there is no antibody loss for marking operations. Since no special equipment is needed, costs are reduced. The activation of the antibodies is not reduced. Since bispecific antibodies are used, the antigen-antibody reaction provides improved reactivity and sensitivity.
In the detection method of the present invention, a fluid sample is applied to a spreading layer from the detection apparatus described above. The bispecific antibodies and the marker are put in a soluble state (which includes states where fine particles are dispersed), where they are allowed to move through the spreading layer. The first bispecific antibody and the second bispecific antibody bond so as to have the detectable material interposed. The first bispecific antibody is bonded to the marker. The second bispecific antibody is bonded to the catcher. At the detection zone, detection results are produced in an amount corresponding to the amount of the detection material in the fluid sample.
With this structure, detection is completed simply by applying the fluid sample, waiting for a fixed period of time, and viewing the detection results at the detection zone.
The detection apparatus according to a feature of the present invention includes a fluid application section contacting a fluid sample. A reaction reagent section connects to the fluid application section. A porous carrier connects to the reaction reagent section. The reaction reagent section includes particles not affecting detection and marking elements. The marking elements bond, through a biochemical reaction, to the particles and the detectable material when the detectable material is present. The particles and the marking elements are movably contained in the porous carrier. A reaction product, produced from bonding between the particles and the marking elements to the detectable material, prevents chromatographic movement of the reaction product. A catching section is disposed at a detection zone to allow chromatographic movement of the marking elements not bonded to the particles. The pore size of the catching section is smaller than the particle diameter of the reaction product and is larger than the particle diameter of the marking element not bonded to the particles.
With this structure, the unmarked reagent is not solidified in the porous carrier and is simply contained in the reaction reagent section. Thus, the amount of unmarked reagent is not restricted by the need to perform solidification. This allows a greater amount of unmarked reagent to be used compared to the conventional detection apparatus, providing improved detection sensitivity. Furthermore, since the unmarked reagent is not physically bound to the porous carrier, the unmarked reagent moves freely through the porous carrier and the free motion (collisions) between components efficiently promotes and speeds up the reaction compared to the conventional technology. This provides improved detection.
Also, if the detectable material is present, the marker bonds with the particles to form a reaction product, which is then caught at the catching section. Thus, there is no hindrance in obtaining the detection results.
Furthermore, the relationship between the pore size and the particle diameter allows the reaction product produced by the bonding between the marking element and the particles to stop and be caught at the catching section.
In the detection apparatus according to another feature of the present invention, the pore size of the catching section is smaller than the particle diameter of the particle.
With this structure, unbonded particles are also stopped at the catching section. This relationship between the diameters can be used when the detectable material has only one immunological reaction site of the same type. In this case, the reaction product and a single particle have roughly the same size, allowing the reaction product to stop at the catching section.
In the detection apparatus according to a further feature of the present invention, the pore size of the catching section is larger than the particle diameter of the particle.
With this structure, unbonded particles pass through the catching section. The relationship between the diameters is suitable for when the detectable material has multiple immunological reaction sites of the same type. In such cases, the detectable material bonds with multiple particles so that the reaction product develops into an aggregate that is much larger than a single particle. Unless the particles are made smaller, the aggregate will become so large that the reaction products can get caught in the middle of the porous carrier without reaching the catching section. Therefore, the particle diameter should be made small to allow the reaction product to chromatographically move to the catching section.